Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

UNLABELLED: To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. IMPORTANCE: Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

Virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cell-mediated capture and transfer of HIV-1 and henipavirus.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63(+)/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination. IMPORTANCE: Dendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive infection in DCs, a mechanism of HIV-1 trans infection. We have recently identified CD169-mediated recognition of GM3, a host-derived glycosphingolipid (GSL) incorporated into the virus particle membrane, as the receptor and ligand for the DC-HIV trans infection pathway. In this study, we have identified the matrix (MA) domain of Gag to be the viral determinant that governs incorporation of GM3 into HIV-1 particles, a previously unappreciated function of the HIV-1 MA. In addition, we demonstrate that the GSL-CD169-dependent trans infection pathway is also utilized as a dissemination mechanism by henipaviruses. GSL incorporation in henipaviruses was also dependent on the viral capsid (M) protein-directed assembly and budding from GSL-enriched lipid microdomains. These findings provide evidence of a conserved mechanism of retrovirus and henipavirus parasitization of cell-to-cell recognition pathways for systemic virus dissemination.

Gag-dependent enrichment of HIV-1 RNA near the uropod membrane of polarized T cells.

The enrichment of HIV-1 macromolecules at the uropod of polarized T cells can significantly promote virus assembly and cell-mediated infection. Using live-cell fluorescence microscopy, we demonstrate that full-length HIV-1 RNA is enriched at the uropod membrane; furthermore, the presence of HIV-1 Gag containing a functional nucleocapsid domain is necessary for this HIV-1 RNA enrichment. The results from these studies provide novel insights into the mechanism of HIV-1 replication in polarized T cells.

Glycosphingolipid composition of human immunodeficiency virus type 1 (HIV-1) particles is a crucial determinant for dendritic cell-mediated HIV-1 trans-infection.

Interactions of human immunodeficiency virus type 1 (HIV-1) with dendritic cells (DCs) are multifactorial and presumably require nonredundant interactions between the HIV-1 envelope glycoprotein gp120 and molecules expressed on the DC surface that define the cellular fate of the virus particle. Surprisingly, neutralization of HIV-1 gp120-dependent binding interactions with DCs was insufficient to prevent HIV-1 attachment. Besides gp120, HIV-1 particles also incorporate host cell-derived proteins and lipids in their particle membrane. In this study, we demonstrate a crucial role for host cell-derived glycosphingolipids (GSLs) for the initial interactions of HIV-1 particles with both immature and mature DCs. Production of HIV-1 particles from virus producer cells treated with ceramide synthase inhibitor fumonisin B1 or glucosylceramide synthase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) resulted in the production of virus particles that, although capable of binding previously defined HIV-1 gp120-specific attachment factors CD4, DC-SIGN, and syndecans, were attenuated in their ability to be captured by both immature and mature DCs. Furthermore, GSL-deficient HIV-1 particles were inhibited in their ability to establish productive infections in DC-T-cell cocultures. These studies provide initial evidence for the role of HIV-1 particle membrane-associated GSLs in virus invasion of DCs and also provide additional novel cellular targets, GSL biosynthetic pathways and GSL-dependent HIV-1 interactions with DCs, for development of antiviral therapy.

Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway.

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.